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Loss of primary sensory neurons in the very old rat Neuron number estimates using the disector method and confocal optical sectioning

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THE JOURNAL OF COMPARATIVE NEUROLOGY 396:211–222 (1998)
Loss of Primary Sensory Neurons in the
Very Old Rat: Neuron Number Estimates
Using the Disector Method and Confocal
Optical Sectioning
E. BERGMAN* AND B. ULFHAKE
Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden
ABSTRACT
Loss of neurons has been considered to be a prime cause of nervous disturbances that
occur with advancing age. However, the notion of a constitutive aging-related loss of neurons
has been challenged recently in several studies that used up-to-date methods for counting
neurons. In this study, we have applied stereological techniques with the objective of obtaining
quantitative data on total neuron numbers and the distribution of neuron cross-sectional
areas in the fifth cervical (C5) and fourth lumbar (L4) dorsal root ganglion (DRG) of 3- and
30-month-old Sprague-Dawley rats. Tissue data were recorded on a confocal laser-scanning
microscope with the use of the optical-disector technique and random, systematic sampling.
Aged rats of both sexes disclosed only a small decrease (<12%) in the number of cervical
and lumbar DRG neurons. Furthermore, there was no significant correlation between the
degree of neuron loss and the extent of behavioral deficits among the aged individuals. The
DRG neurons of aged rats had a smaller mean cross-sectional area (<15%; P , 0.001) at both
DRG levels. Further analysis of the male cohorts was carried out by using isolectin B4 and
neurofilament subunit (phosphorylated 200 kDa; RT97) immunoreactivity (IR) as selective
markers for unmyelinated and myelinated axons, respectively, and disclosed no significant
change in the relative frequencies of immunoreactive neuron profiles in the old rats. However,
RT97-IR DRG neurons of the aged rats had significantly smaller cross-sectional areas (<9% in
C5; <16% in L4; P , 0.001) than the young adult rats, indicating a selective cell body atrophy
among myelinated primary afferents during aging.
The results indicate that loss of primary sensory neurons cannot exclusively explain the
functional deficits in sensory perception among senescent individuals. It seems likely that
other factors at the subcellular level and/or target interaction(s) contribute substantially to
the sensory impairments observed with advancing age. J. Comp. Neurol. 396:211–222,
1998. r 1998 Wiley-Liss, Inc.
Indexing terms: aging; stereology; optical disector; dorsal root ganglia; spinal cord
Aging is associated with neurological symptoms and
signs that are suggestive of peripheral neuropathy (Critchley, 1956; Cavanagh, 1964; Jellinger, 1973; Kokmen et al.,
1977). Thus, increased thresholds for tactile, thermal, and
vibratory sensations are common in senescent mammals
(Dyck et al., 1984; de Neeling et al., 1994; Gescheider et al.,
1994; Bergman et al., unpublished observations), and
studies of sensory nerves in aged animals have shown
axonal dystrophy, demyelination, and axon degeneration
(Berg et al., 1962; van Steenis and Kroes, 1971; Gilmore,
1972; Burek et al., 1976; Thomas et al., 1980; CotardBartley et al., 1981; Mitsumori et al., 1981; Mittal and
Logmani, 1987).
Loss of neurons during senescence, which has long been
considered to be an important trait of aging, is currently
r 1998 WILEY-LISS, INC.
the subject of reexamination (for review, see Wickelgren,
1996). Earlier studies of neuron loss during aging were
often compromised by inconsistent results (for review, see
Coleman and Flood, 1987). For example, results from
Grant sponsor: Swedish Medical Research Council; Grant number:
10820; Grant sponsor: Karolinska Institutet; Grant sponsor: L and H
Ostermans Fond för Medicinsk Forskning; Grant sponsor: A and M
Wallenbergs Minnesfond; Grant sponsor: Kapten A. Erikssons Stiftelse för
Medicinsk Forskning.
*Correspondence to: Esbjörn Bergman, Department of Neuroscience,
Karolinska Institute, S-171 77 Stockholm, Sweden.
E-mail: esbjorn.bergman@neuro.ki.se
Received 6 August 1997; Revised 29 December 1997; Accepted 1 January
1998
212
E. BERGMAN AND B. ULFHAKE
studies of dorsal root ganglia (DRGs) are at variance, with
some revealing a loss of up to one-third of the neurons with
advancing age (Gardner, 1940; Nagashima and Oota,
1974), whereas others show no reduction in cell numbers
(Emery and Singhal, 1973; Ohta et al., 1974; La Forte et
al., 1991). A problem with all of these studies is that they
were performed by using indirect techniques based on
two-dimensional images or probes, thereby producing biased results (e.g., see Coggeshall, 1992). With the development of stereological techniques (for reviews, see Sterio,
1984; Gundersen et al., 1988a,b), it seems possible to
circumvent these methodological problems in producing
accurate estimates of neuron numbers.
Since a precise knowledge about the morphometric
characteristics of DRGs may help us to understand some
aspects of the functional deficits occurring with advancing
age, we have applied stereological techniques to estimate
the total number of neurons and their cross-sectional
areas in cervical and lumbar DRGs of young adult (3
months old) and aged (30 months old) Sprague-Dawley
rats. Both males and females were included to examine
any possible difference(s) related to sex. Furthermore, a
distribution analysis, with immunoreactivity for the phosphorylated 200-kDa neurofilament subunit (RT97) and
isolectin B4 as markers for large light (myelinated; Lawson et al., 1984) and small dark (unmyelinated; Wang et
al., 1994) DRG neurons, respectively, was performed in
order to shed some light on any possible selectivity of aging
on subpopulations of DRG neurons.
MATERIALS AND METHODS
Experimental animals and
behavioral deficits in aged rats
In this study, male and female Sprague-Dawley rats
(strain Bkl; Harlan Sprague-Dawley, Houston, TX), including a total of nine young adult (2–3 months old; body
weight: male 300–400 g, n 5 4; female 200–250 g, n 5 5)
and eight aged rats (30 months old; body weight: male
590–700 g, n 5 3; female 290–400 g, n 5 5), were used.
The animals were delivered by a local breeder (B and K,
Stockholm, Sweden) at 2 months of age and were kept
thereafter under standardized barrier-breeding conditions
at our department (12-hour light/12-hour dark cycle) with
free access to water and food (R70 with reduced protein
content; Lactamin, Vadstena, Sweden). Under these conditions, the median life span is about 30 months (62 months
across cohorts) for both males and females (see also
Gutman and Hanzlikova, 1972; Burek and Hollander,
1980; Masoro, 1980; Algeri et al., 1983). Based on this, the
30-month-old rats were defined as ‘‘aged.’’
Rats show a progressive deterioration of motor behavior
during aging (Berg et al., 1962; van Steenis and Kroes,
1971; Burek et al., 1976; Mitsumori et al., 1981; Krinke,
1983; Johnson et al., 1993), with symptoms usually starting during the third year of life and affecting mainly the
hindlimbs (‘‘posterior paralysis’’). All aged rats used in this
study disclosed signs of behavioral sensory-motor disturbances, defined according to a previously described staging
protocol (Johnson et al., 1995). Briefly, the symptoms were
most evident in the hindlimbs and ranged from a moderate
muscular atrophy and an adduction insufficiency in the
least affected cases (stage I) to a more or less complete
paralysis of the hindlimbs with severe wasting of the
hindlimb muscles in the most severely affected animals
(stage III). Among the aged rats used in this study, all
three stages were represented.
Behavioral testing of sensory functions included nociceptive (hot-plate test; Espejo and Mir, 1993; Langerman et
al., 1995) and tactile (von Frey test; Tal and Bennett, 1994)
thresholds and will be described in detail elsewhere (Bergman et al., unpublished observations). Briefly, nociceptive
thresholds (response latency) were increased in aged rats
(see Fig. 1A), and there was a positive correlation between
response latency and age/stage (Fig. 1A). Also, tactile
thresholds increased significantly from 3 month to 30
months of age (only females tested; Fig. 1B). In the latter
test, however, no consistent correlation to clinical stage
could be revealed among the aged rats. All experiments
were approved of by the Local Ethical Committee (Stockholm’s Norra Djurförsöksetiska Nämnd; project no. 263/
95).
General procedures
The rats were deeply anaesthetized with chloral hydrate
(300 mg kg21, i.p.) and perfused transcardially with warm
(37°C), Ca21-free Tyrode’s solution followed by cold fixative
(4°C) containing 4% w/v paraformaldehyde and 0.2% w/v
picric acid in 0.1 M phosphate-buffered saline (PBS), pH
7.2; Pease, 1962; Zamboni and De Martino, 1967) for 8
minutes. The fifth cervical (C5) and fourth lumbar (L4)
DRGs were dissected out, immersed in fresh fixative for 90
minutes, and then stored in 10% sucrose buffer solution
containing 0.01% sodium azide (Sigma, St. Louis, MO) and
0.02% Bacitracin (Bayer, Leverkusen, Germany) overnight
at 4°C. Complete section series of the DRGs randomly
orientated around their longitudinal axis were cut at 14
µm in a cryostat (Dittes, Heidelberg, Germany) and thaw
mounted onto chrome-alum gelatin-coated slides.
Tissue preparation
Conventional histochemical procedures. With a random starting point, sections to be used for cell counting
were systematically sampled throughout the complete
section series and stained with a fluorescent counterstain.
Briefly, the air-dried sections were rehydrated in 0.1 M
PBS, counterstained for 5 minutes in 0.001% (w/v) propidium iodide (Sigma) in PBS, rinsed three times for 5
minutes each in PBS, and mounted in glycerol. Propidium
iodide, which is known to bind to nucleic acids, yielded a
pattern that closely resembled conventional Nissl staining, with fluorescent brightly cytoplasm and nucleoli.
Immunohistochemical procedures. Remaining sections from male young adult and aged rats were used for
B4 or RT97 immunohistochemistry according to the indirect immunofluorescence technique of Coons and collaborators (see Coons, 1958). After rehydration in 0.1 M PBS, the
sections were preincubated for 1 hour at room temperature
in PBS containing 10% normal donkey serum and 0.3%
Triton X-100.
For B4 immunohistochemistry, sections were incubated
with the isolectin B4 from Griffonia simplicifolia I (B4; 10
µg/ml; Vector Laboratories, Burlingame, CA) for 18 hours
at 4°C. The sections were then rinsed three times for 10
minutes each in PBS and incubated with goat antiGriffonia simplicifolia lectin I (GSA I) antiserum (1:1,000;
Vector Laboratories) for 18 hours at 4°C. For RT97 immunohistochemistry, sections were incubated with a mouse
anti-RT97 monoclonal antibody (phosphorylated 200 kDa
NEURON LOSS IN AGED RAT DRG
213
Fig. 1. Effects of aging on nociceptive (hot-plate test; A) and tactile
(von Frey test; B) thresholds. The aged rats (stages I–III) disclosed
significantly higher thresholds for both nociceptive and tactile stimuli
than the young adult rats (analysis of variance [ANOVA], P , 0.001).
In the hot-plate test, there was a positive correlation between response
latency and age/stage (Spearman correlation coefficient 5 0.59;
P , 0.001), whereas, in the von Frey test, no such correlation was
found among the aged rats. Each point represents the mean 6
standard error of mean (S.E.M.). Levels of significance are described in
Materials and Methods under Statistics.
neurofilament (1:250, kindly provided by J.W. Wood; Wood
and Anderton, 1981) for 72 hours at 4°C.
After incubation with the primary antibodies, the sections were thoroughly rinsed in PBS and incubated with
dichlorotriazinyl aminofluorescein (DTAF)-conjugated donkey anti-goat IgG (1:40; Jackson Immuno Research, West
Grove, PA) or DTAF-conjugated donkey anti-mouse IgG
(1:40; Jackson Immuno Research) for 30 minutes at 37°C.
After a final rinse in PBS, the sections were mounted in
glycerol/PBS (3:1) containing 0.1% r-phenylenediamine in
order to retard fading (Johnson and de C Nogueira Araujo,
1981; Platt and Michael, 1983). All antisera/antibodies
were diluted in 0.1 M PBS containing 1% bovine serum
albumin, 0.3% Triton X-100, 0.01% sodium azide, and
0.02% Bacitracin.
DRG volume. The Cavalieri method (Gundersen and
Jensen, 1987) was used to estimate the volume of the DRG.
With a random starting point, 10–15 equally spaced
sections through the ganglia were analyzed. Each section
was recorded with a 43 objective (5-µm pixel spacing), and
the cross-sectional area of each ganglion section was
measured by using Image Space software (Fig. 2A). For
measurements of section thickness, axial (z-)-scans
(0.10-µm step size) were sampled in a random systematic
fashion across each section with a 403 objective (Fig. 2B).
With the used rehydration and embedding protocol (see
above), the tissue shrinkage was negligible (Fig. 2B). Tests
with ethanol dehydration and embedding in Entellan
(Merck, Darmstadt, Germany) resulted in a 30–50% shrinkage of the tissue sections (data not shown). The total
ganglion volume was calculated by multiplying the mean
cross-sectional area, the mean section thickness, and the
number of sections.
Neuron density. The optical-disector principle (Gundersen, 1986; Gundersen et al., 1988a) was applied on the
sections used for the Cavalieri estimates. The sampling
scheme employed was based on the results from a pilot
study in which the appropriate number of disectors was
determined. In total, approximately 65 disectors were
analyzed in each ganglion. With a random starting point in
each section, visual fields were sampled systematically by
using an x-y-axis step size of 600 µm or 800 µm for the fifth
cervical vertebrae (C5) and the fourth lumbar vertebrae
(L4) DRGs, respectively. At each sample location, optical sectioning was performed with the CLSM by using a 403/1.0
NA planApo oil-immersion objective with an axial resolution of 2.5 µm (defined by the full-width half maximum;
Stereology
To obtain an unbiased estimate of the total number of
neurons, we used the disector principle and random systematic sampling (Sterio, 1984; Gundersen et al., 1988a; West,
1993). The analysis was performed on a Sarastro 1000
(Molecular Dynamics, Inc., Sunnyvale, CA) confocal laserscanning microscope (CLSM) with laser wave lengths and
filters set for propidium iodide fluorescence. Briefly, the
514-nm line of an argon-ion laser was used as the excitation light, and the propidium iodide fluorescence emission
light was collected through a dichroic mirror (split wave
length 525 nm) and a long-pass filter (LP 530 nm). Images
were recorded with either a 403/1.0 oil-immersion planapochromate objective or a 43 air objective and stored in a
computer for subsequent analysis in Image Space software
(Molecular Dynamics, Inc.).
214
E. BERGMAN AND B. ULFHAKE
Figure 2
NEURON LOSS IN AGED RAT DRG
see Ulfhake et al., 1994). The starting point, which was
derived from a z-scan, was set 2 µm below the section
surface. From this point, nine consecutive optical sections
were recorded by using a z-axis step size of 1 µm. The
resulting stack of digital images, with a lateral pixel
spacing of 0.25 µm, was analyzed on a computer equipped
with the Image Space software. An unbiased counting
frame with an area of 39,828 µm2 was presented by the
computer, and all neurons with a nucleolus in the starting
plane (i.e., optical section one) or in the guard volume,
which includes the immediately adjacent mechanical section, were disregarded. In the following eight sections, i.e.,
section two through nine, all neurons with a distinct
nucleolus appearing inside the counting frame were
counted (Fig. 2C–K). In this way, by using optical disectors
with a height of 8 µm, an average of 165 neurons were
sampled in each ganglion. The neuron density was then
calculated as the sum of all neurons counted divided by the
summed volume of all disectors.
Neuron numbers and cross-sectional areas. The
total number of neurons was calculated as the product of
the volume and the numerical density, i.e., the number of
neurons per unit volume, in each ganglion. All cells
sampled during the stereological procedure had their
cross-sectional area measured in the plane of the nucleolus. The measurements were performed on the digitized
images, and the results were stored in the computer for
subsequent analysis.
Quantitative evaluation of B4- and
RT97-immunoreactive neuron profiles
To determine the relative frequencies and size distributions of B4- and RT97-immunoreactive (IR) neuron profiles
in C5 and L4 DRGs, eight to ten sections were sampled
systematically throughout each ganglion in both young
adult and aged male rats. In each section, two to four
randomly selected fields were collected by using a Nikon
microscope (Tokyo, Japan), with a 203/0.75 NA objective
lens equipped with a Ultrapix 1600 CCD camera (AstroCam Ltd., Cambridge, United Kingdom). The digital images, each comprising 1,536 3 1,024 pixels with 8 bits of
Fig. 2. A–K: Confocal images illustrating the principles of the
optical disector method used for quantification of total neuron numbers and cross-sectional areas. A: Overview, which was scanned with a
43 objective, of a randomly, systematically sampled section in a fourth
lumbar vertebrae (L4) dorsal root ganglion (DRG) of a young adult
male rat. Within each selected section, a random starting point was
chosen, and fields were then sampled systematically and analyzed
(squared area in A). The overview image is also used to measure the
cross-sectional area of the DRG section for the estimation of total
ganglion volume, according to the Cavalieri principle. B: A z-scan,
which was captured with a 403/1.0 NA plan-Apo oil-immersion
objective, revealing a section thickness of 14 µm. C–K: Confocal optical
sections, which were scanned with a 403/1.0 NA plan-Apo oilimmersion objective, through the tissue specimen. Starting 2–3 µm
below the section surface, as decided from the preceding z-scan, nine
consecutive images were recorded by using a step size of 1 µm. All
neurons containing a nucleolus in the first section (arrowheads in C)
were disregarded. In the following eight sections (D–K), all neurons
with a distinct nucleolus (solid arrows in I and K) that appeared
within the unbiased counting frame (square in C–K) were counted and
had their cross-sectional area measured. Nucleoli touching the upper
or right border of the counting frame were considered to be inside,
whereas those touching the lower or left border (open arrow in I) were
considered to be outside and, hence, were not counted. Scale bars 5
200 µm in A, 5 µm in B, 20 µm in C (also applies to D–K).
215
data, were analyzed by using the Optimas 6.0 software
(Optimas Corporation, Bothell, WA) to calculate the ratios
of labeled/unlabeled neuron profiles. Only cell profiles
containing a clearly visible nucleus were included. All
immunopositive neuron profiles also had their crosssectional area measured in the nuclear plane. In total,
830–1,280 profiles were analyzed in each DRG.
Statistics
To analyze the appropriateness of the sampling scheme
employed, the coefficient of variance (CV; S.D./mean) and
the coefficient of error (CE) were determined. The CE of
the individual estimates, reflecting the precision of the
sampling procedure, was calculated according to West and
Gundersen (1990; see also Gundersen and Jensen, 1987).
Analysis of variance (ANOVA) with Fisher’s LSD was
used 1) to test for the effects of age and sex, respectively, on
total neuron numbers at the two DRG levels studied; and
2) to evaluate the effect of aging on the relative frequency
and size distribution of neuron profiles immunoreactive for
B4 and RT97 in C5 and L4 DRGs.
When analyzing the cell-size distribution, nonparametric Kruskal-Wallis one-way ANOVA and contingency table
analysis (x2; bin data) were used. For contingency table
analysis, each set of data was divided into three size bins
corresponding to small (,750 µm2), medium (750–1,750
µm2), and large (.1,750 µm2) DRG neurons, respectively
(see also Rambourg et al., 1983; Price, 1985; Tandrup,
1993; Zhang et al., 1994). In the histograms, the levels of
significance are been indicated as follows: n.s., nonsignificant; single asterisk, P , 0.05; double asterisks, P , 0.01;
triple asterisks, P , 0.001 (see Figs. 1, 3).
RESULTS
Numbers of neurons
The numbers of neurons in C5 and L4 DRGs of young
adult and aged rats of both sexes are indicated in Figure 3.
Aged animals, on the average, had 12% fewer cells in both
C5 (11.7–13.9%) and L4 (10.8–13.5%) DRGs, with no difference in the degree of cell loss between male and female
rats. The decrease in neuron numbers with age was highly
significant for female L4 DRGs (ANOVA; P , 0.001) and, to
a lesser extent, also for cervical ganglia of both sexes
(ANOVA; P 5 0.04 and P 5 0.02 for male and female rats,
respectively). The small difference between young adult
and aged male L4 DRGs was not significant (ANOVA;
P 5 0.11). No correlation was found between the degree of
cell loss and the clinical symptoms (stage) of the rats
(Fig. 4).
Both young adult and aged male rats showed approximately 6% fewer cells than female rats in both cervical and
lumbar DRGs. However, these differences were not statistically significant (ANOVA; P 5 0.09–0.26).
Aged rats, of both sexes, had an increased DRG volume
of approximately 16% (C5) and 26% (L4). In parallel, a
consistent reduction in the neuron density was observed.
Analysis of the sampling scheme revealed that the mean
observed relative variation among animals, coefficient of
variation (CV2), was 0.0087, and the average coefficient of
error (CE2; see Materials and Methods) of the estimates
was 0.0024, with an equal contribution of the estimates for
ganglion volume and numerical density. Thus, the biological variance contributed more than two-thirds of the total
variance.
216
E. BERGMAN AND B. ULFHAKE
Fig. 3. Graphic representation of the total number of neurons in
the fifth cervical (C5) and fourth lumbar (L4) dorsal root ganglia (DRG)
in young adult (open symbols) and aged (solid symbols), male (squares)
and female (circles), Sprague-Dawley rats. Horizontal bars represent
mean values. Levels of significance are described in Materials and
Methods under Statistics.
Fig. 4. Graphic representation of the relation between the total
number of neurons in C5 (below hatched line) and L4 (above hatched
line) dorsal root ganglia (DRG) and age (young adult, open symbols;
aged stages I–III, solid symbols). Male and female rats are indicated
with squares and circles, respectively. Note the lack of correlation
between neuron loss and symptoms among the aged rats at both DRG
levels studied.
TABLE 1. Mean Cross-Sectional Areas in C5 and L4 DRGs of Young Adult
(Female and Male) and Aged (Female and Male) Rats1
C5 cross-sectional area µm2 (mean 6 S.D.)
L4 cross-sectional area µm2 (mean 6 S.D.)
Cell size distribution
Cell body cross-sectional areas in cervical and lumbar
DRGs of both female and male aged rats were found to be
approximately 15% smaller than in young adult rats
(Table 1, Fig. 5). The difference in cell size distribution
between young adult and aged rats was statistically
significant (Kruskal-Wallis; P , 0.001) in both C5 and L4
DRGs. Comparison of male and female rats within age
groups revealed no statistically significant difference
(Kruskal-Wallis; P 5 0.06–0.57).
Figure 5 also illustrates the frequency of neurons in the
three size categories used to define small, medium, and
large DRG neurons. The histograms clearly indicate a shift
toward smaller cell categories among the neuron populations of aged animals. A significant difference was evident
in both C5 and L4 DRGs when comparing young adult and
aged rats (x2; P , 0.001) of both sexes, whereas no difference was seen between males and females within the age
groups (x2; P 5 0.08–0.55).
Examination of B4- and RT97-IR neurons
The relative distribution of B4 and RT97 immunoreactivity (Fig. 6) was examined in the male age cohorts to
1C
5,
Young adult
(n 5 9)
Aged
(n 5 8)
926 6 592
1,165 6 809
784 6 472
993 6 614
fifth cervical vertebrae; L4 , fourth lumbar vertebrae; DRGs, dorsal root ganglia.
determine whether the observed reduction in cell-number
and cell-size in aged rats, showed any selectivity for
non-myelinated or myelinated DRG neurons. In Table 2,
the relative frequencies of B4- and RT97-IR profiles in
young adult and aged rat DRGs have been tabulated. The
data show that the relative proportions of the two neuron
populations at both DRG levels remain virtually unchanged during aging (ANOVA; P 5 0.27–0.94).
Examination of the distribution of cross-sectional areas
of B4-IR neuron profiles showed that the mean area in the
aged rats was approximately 2% smaller than in the young
adult rats in both C5 and L4 DRGs. However, among
RT97-IR neuron profiles, 9% (ANOVA; P , 0.001) and 16%
(ANOVA; P , 0.001) decreases in mean cross-sectional
area were found in C5 and L4 DRGs, respectively, of the
aged rats. The difference was most prominent for L4 DRGs,
in which all aged rats were significantly different from the
young adult rats, whereas, in C5 DRGs, the aged rats
differed significantly from two of the young adult rats.
Thus, cell body atrophy in aged rats was only recorded in
the myelinated subpopulation of DRG neurons.
NEURON LOSS IN AGED RAT DRG
217
Fig. 5. Size frequency histograms illustrating the distribution of
C5 (A) and L4 (B) dorsal root ganglia (DRG) neurons in young adult
(male 1 female; gray) and aged (male 1 female; black) rats. The
frequency (6S.D.) of neurons that belong to each of the three size bins
(indicated by vertical hatched lines), as defined in Materials and
Methods, is indicated for both age groups. Note the shift toward
smaller cells among the aged rats.
DISCUSSION
Changes in cell body cross-sectional areas seem to implicate a selective atrophy of large myelinated primary
afferents during aging (see also below). However, our data
do not suggest that neuron loss in DRGs was more
extensive among any subpopulation of primary afferents.
This study shows that aging is associated with only a
small loss of DRG neurons, equally evident at cervical and
lumbar levels, in Sprague-Dawley rats of both sexes.
218
E. BERGMAN AND B. ULFHAKE
Fig. 6. Immunofluorescence images of young adult (A,C) and aged
(B,D) male rat L4 dorsal root ganglia (DRGs) after incubation with B4
(A,B) and RT97 (C,D). With regard to frequencies of immunopositive
neuronal profiles, no difference could be observed when comparing
young adult and aged rats. However, for RT97, but not for B4, a
significant decrease in mean cross-sectional area could be observed in
the aged compared with the young adult rats. Small (A,B), medium
(A–D), and large (C,D) arrows point to small, medium-sized, and large
DRG neurons, respectively. Scale bar 5 70 µm.
TABLE 2. Frequencies and Mean Cross-Sectional Areas of B4- and RT97Immunoreactive neurons in C5 and L4 DRGs of Young Adult and Aged Rats
and confocal microscopy. In contrast to conventional microscopy, confocal microscopy provides a resolution along the
optical axis (for references, see Ulfhake et al., 1994),
enabling true optical sectioning of tissue specimens. Thus,
recording of profiles in focus is operator independent and is
therefore unbiased. We have chosen to count a neuron the
first time that a nucleolus appears in the profile, which is a
unique event. Quite frequently, multiple nucleoli were
encountered, especially in the smaller type-B cells. To
secure the validity of the counting, the tissue volume above
the starting plane (i.e., guard volume), including the
immediately adjacent mechanical section, was checked for
possible occurence of multiple nuclei/nucleoli in the counted
cell. Even though the methodology employed here is
considered insensitive to tissue shrinkage with regard to
total neuron numbers, shrinkage will affect the crosssectional area measurements and, moreover, will increase
the demand on the optical depth resolution. To circumvent
the problem with tissue shrinkage, observed in, e.g.,
paraffin sections (see Schmalbruch, 1987), the sections
were rehydrated in PBS and embedded in glycerol, which
preserves the tissue volume. In test experiments with
tissue embedding in Entellan (Merck) following dehydra-
C5
Young
adult
(n 5 4)
B4
Frequency (% 6 S.D.)
Cross-sectional area
µm2 (mean 6 S.D.)
RT 97
Frequency (% 6 S.D.)
Cross-sectional area
µm2 (mean 6 S.D.)
L4
Aged
(n 5 3)
Young
adult
(n 5 4)
Aged
(n 5 3)
51 6 7
51 6 5
50 6 5
50 6 7
612 6 157
587 6 131
726 6 199
718 6 181
43 6 7
44 6 8
44 6 8
46 6 7
1,384 6 467
1,266 6 412
1,923 6 590
1,612 6 511
Furthermore, there were no indications of a correlation
between neuron loss and the degree of clinically manifest
dysfunction among the aged rats.
Methodological considerations
To our knowledge, this is the first attempt to quantify
changes in DRGs of both sexes during aging by using
stereological techniques. Here, we have employed the
disector principle (Sterio, 1984; Gundersen et al., 1988a)
NEURON LOSS IN AGED RAT DRG
tion, we recorded a reduction of the section thickness by
approximately 30–50%.
It was calculated that the biological variance contributed more than two-thirds of the total variance in each age
group and spinal cord level, which is satisfactory, in that
the stereological approach contributes only a minor fraction of the observed variance.
Number of DRG neurons and
loss of neurons during aging
Our values for total number of L4 DRG neurons in young
adult rats are close to those obtained with the same
technique in rat L5 DRGs (Tandrup, 1993). Earlier estimates using other counting methods have shown a severalfold difference in neuron numbers across studies (Bondok
and Sansone, 1984; Feringa et al., 1985; Arvidsson et al.,
1986), and this inconsistency has been attributed to imperfection(s) in the applied counting procedures (Coggeshall
et al., 1990; Pover and Coggeshall, 1991). Furthermore,
with the exception of a complete serial section study of
DRGs (Schmalbruch, 1987), neuron counts in the studies
cited above have yielded much lower values than those
obtained with the disector technique.
Examination of neuron number in DRGs during aging
has produced inconsistent results (Gardner, 1940; Emery
and Singhal, 1973; Nagashima and Oota, 1974; Ohta et al.,
1974; La Forte et al., 1991), as discussed above, and this
variation is most likely related to the counting procedures
used. The results obtained here show that loss of primary
sensory neurons is small also in the very old rat and,
furthermore, that there was no discernible difference in
the degree of cell loss between sexes or between levels of
the spinal cord. The findings implicate that cell loss itself
cannot account for the sensory deficits seen in elderly
individuals. Furthermore, our results are consistent with
studies of dorsal roots that have described only small or no
loss of (myelinated) fibers with advancing age (Mitsumori
et al., 1981; Rao and Krinke, 1983; Knox et al., 1989). The
present findings, moreover, add to the growing body of
studies showing that aging is not accompanied by a
substantial loss of neurons (see e.g., Satorre et al., 1985;
Ahmad and Spear, 1993; Madeira et al., 1995; for reviews,
see Coleman and Flood, 1987; Wickelgren, 1996).
Aged rats
In this study, the 30-month cohort was used as ‘‘aged.’’ In
the literature, the median survival age of rats varies
between 26 months and 32 months (Gutman and Hanzlikova, 1972; Burek and Hollander, 1980; Masoro, 1980;
Algeri et al., 1983; Johnson et al., 1993, 1995; Hashizume
and Kanda, 1995). Outbred Sprague-Dawley rats kept in a
barrier facility, on average, reach an age of about 30
months, and, as described here and elsewhere, litter mates
at this age disclose highly variable degrees of nervous
function incapacitations, reflecting differences among the
individuals (Bergman et al., unpublished observations; see
also van Steenis and Kroes, 1971; Burek et al., 1976;
Johnson et al., 1995). Although the number of DRG
neurons varied among the aged rats, this variation did not
appear to be linked to the extent of sensory-motor dysfunction of the individuals, to sex, or to the level of the spinal
cord.
219
Sensory impairments and selective
vulnerability during aging
With advancing age in both rodents and humans, exteroception and proprioception become impaired (Foster et al.,
1976; Macintosh and Sinclair, 1978; Kenney and Fowler,
1988; Schmidt et al., 1990; Ferrell et al., 1992; AbdelRahman and Cowen, 1993; de Neeling et al., 1994; Gescheider et al., 1994; Ferrer et al., 1995; Quoniam et al.,
1995; Robbins et al., 1995). Peripheral nerves of aged
animals show both loss of fibers and degenerative changes
(Cowen et al., 1982; Dhall et al., 1986; Mione et al., 1988;
Cowen and Thrasivoulou, 1990; Navarro and Kennedy,
1990; Abdel-Rahman and Cowen, 1993). These data are
consistent with studies of sensory nerves and dorsal roots
demonstrating axonal dystrophy, demyelination, and axon
degeneration as well as loss of fibers during aging (Berg et
al., 1962; van Steenis and Kroes, 1971; Gilmore, 1972;
Burek et al., 1976; Sharma et al., 1980; Thomas et al.,
1980; Cotard-Bartley et al., 1981; Mitsumori et al., 1981;
Krinke, 1983; Mittal and Logmani, 1987; Knox et al.,
1989). It seems that peripheral nerves are affected more
than both dorsal roots (Rao and Krinke, 1983; Knox et al.,
1989) and the parent cell bodies in the DRGs.
A considerable loss of dermal innervation has been
described in skin from both rodents and humans (see e.g.,
Abdel-Rahman and Cowen, 1993; Fundin et al., 1997),
implicating poorer thermal and tactile sensibility. This is
consistent with our behavioral tests of aged rats, which
show increases in both hot-plate response latency and von
Frey hair threshold. Furthermore, several studies have
shown that there appear to be specific patterns for sensory
fiber loss during aging (Cauna, 1965; Fundin et al., 1997),
for example, with aging, proprioceptive receptors/sensory
neurons degenerate earlier and more extensively than
nociceptive fibers (Fundin et al., 1997). A differential effect
of aging on tactile skin receptors has also been demonstrated in both rodents and humans (Cauna, 1965; Winkelmann, 1965; Bolton et al., 1966; Schimirgk and Ruttiger,
1980; Cerimele et al., 1990; Fundin et al., 1997). Furthermore, physiological tests of tactile responses in the finger
pulp (Schmidt et al., 1990) have indicated that the distal
axon, including the endings, seems to be affected more
severely than the proximal axon, thus, supporting the
notion that neuronal aging is often a distal-to-proximal
process.
Proprioceptive signals are conveyed most often in Aa
and Ab fibers, whereas nociception travels in Ad and C
fibers. Axon dystrophy, degeneration, and demyelination
in peripheral nerves and spinal roots are particularly
frequent in myelinated, large-diameter fibers (Spencer
and Ochoa, 1981; Krinke, 1983; Rao and Krinke, 1983;
Knox et al., 1989). The stereological data recorded in aged
rat DRGs may be interpreted to show that aging is
associated with a selective loss of large (myelinated) DRG
neurons. However, it cannot be excluded that cell loss and
cell atrophy occur as independent processes during aging
and, hence, that cell loss may be unselective concerning
cell size, whereas, compared with small DRG neurons,
large DRG neurons may be more prone to atrophy. To shed
some light on this issue, we employed immunohistochemistry by using B4 and RT97, respectively, as markers for
unmyelinated and myelinated neurons. This examination
revealed no difference between young adult and aged rats
in relative frequencies of B4- and RT97-IR neuron profiles,
220
E. BERGMAN AND B. ULFHAKE
indicating that there is probably no selective loss of small
or large DRG neurons during aging. However, the RT97-IR
cells, which presumably give rise to large myelinated
axons, showed a pronounced reduction in mean crosssectional area in the nuclear plane. Thus, it may indeed be
that the decrease in neuron size recorded in the stereological part of this study can be explained by a selective
atrophy among RT97-IR neurons. The cell body atrophy
among sensory neurons observed here is consistent with a
previous report (Rao and Krinke, 1983) in which a decreased mean cell size was found in aged rat DRGs.
Several studies have shown a decreased expression of
neurofilaments in aged rat primary sensory neurons
(Parhad et al., 1995; Kuchel et al., 1996), which is interesting, because neurofilaments are the major determinants of
axonal caliber (Hoffmann et al., 1987). A decreased capacity for, in particular, large sensory neurons to sustain their
cytoskeletal framework could be a plausible explanation
for axon dystrophy/cell atrophy. The mechanism behind a
decreased expression of neurofilaments remains unclear.
However, the expression of neurofilaments is believed to be
regulated by a neurotrophic signal from the periphery
(Gold et al., 1991; Parhad et al., 1995), and we have shown
previously that aged rats have a decreased expression of
neurotrophin receptors (Bergman et al., 1996). Clearly,
this issue cannot be resolved here but deserves further
study.
In this context, it seems appropriate also to comment on
the notion that neuropathy in the elderly is caused commonly by mechanical trauma, like nerve entrapment or
disc hernia. However, the characteristic changes in neurons with advancing age are common in the mystacial pad
(Fundin et al., 1997), the nerve to the tail (Thomas et al.,
1980), and systems intrinsic to the central nervous system
(Johnson et al., 1993); furthermore, they also show a high
selectivity within a nerve, making it highly unlikely that
pressure trauma alone is the common etiology to neuropathy among senescent individuals.
CONCLUSIONS
Loss of neurons has long been considered a key element
contributing to the symptomatology observed in elderly
individuals. Our results using confocal microscopy and the
disector technique show that the degree of DRG neuron
loss in aged rats is at a level that is unlikely to explain fully
the functional deficits. Furthermore, the reduction in
neuron numbers did not differ between spinal cord levels
or between sexes; moreover, there was a lack of correlation
between behavioral symptoms and neuron loss among the
aged rats. Finally, we provide some evidence indicating
that aging may be associated with a selective atrophy of
large, myelinated, primary sensory neurons.
ACKNOWLEDGMENTS
We thank Prof. N. Åslund and Prof. K. Carlson for
permission to use the confocal microscopes at Physics 4,
The Royal Technical Institute, Stockholm.
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